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Summarized manual for FLIM experiments
17.12 Measuring the Instrument Response Function (IRF)
For accurate measurements of lifetimes near to the timing resolution, the instrument
response function (IRF) should be taken into account. The width of the IRF displays the
timing resolution of the instrument.
For determination and use of the instrument response function (IRF) in MP systems with HyD
RLD, it is advisable to place a black, non-reflective piece of paper between the specimen
and the condenser.
17.12.1 Preparing IRF Measurements
There are different ways to obtain an IRF.
17.12.1.1 Estimating the IRF
The IRF function normally has to be measured separately. However, SymPhoTime can also
measure the IRF function starting from the ascending edge of the TCSPC decay curve.
17.12.1.2 With Reflection Mode
Only use the reflection mode if no suitable fluorescence specimen is available. It might
create additional signals not relevant in real fluorescence experiments. Aside from that, the
IRF is dependent on the wavelength APD.
1. Position a backscattering specimen (such as a cover slip or a mirror) in the laser focus
on the specimen stage of the microscope. Alternatively, you can use Ludox or milk in a
measurement chamber.
2. Remove all barrier filters from the emission path so that the laser line used reaches the
detector.
3. For external FLIM detection, remove the band pass filters from the SMD filter cube.
4. Bring the fluorifier disc into the
Substrate
position.
5. Place an OD3 attenuating filter in the filter holder in front of the detector or apply very
low laser intensity. The correct filter holder position can be taken from the detector
manual.
Pay attention to the SymPhoTime online help
Further information can be found in the SymPhoTime online help.